Investigating the Role of SMN in Ribosome Heterogeneity

Ribosome heterogeneity is an emerging concept in biology. Despite their conserved core structure, evidence accrued over the years shows that ribosomes may differ at multiple levels: protein and rRNA composition, post-transcriptional and post-translational modifications, and association with proteins and ncRNAs. Together, these levels of heterogeneity can influence transcript selection or ribosome function, favoring the translation of specific subsets of mRNAs. However, the role of ribosome heterogeneity in disease pathogenesis remains elusive. SMN protein is a Ribosome Associated Protein (RAP), which likely contributes to shape ribosome heterogeneity. Decreased levels of SMN protein are responsible for Spinal Muscular Atrophy (SMA), a life-threatening genetic disease which primarily presents neuromuscular manifestations. Recently, translational defects have been identified in SMA. The aim of this work is to further characterize the contribution of SMN in determining ribosome heterogeneity at protein and post-transcriptional level, and to understand the implications of the loss of regulation of translation by SMN in SMA. To identify additional levels of heterogeneity controlled by SMN in ribosomes, I used multiple proteomics analysis, polysome profiling, ribosome profiling complemented with translational assays in in vitro, in cellulo and in vivo models of disease. Through these techniques it was demonstrated: i) the existence of putative binding sites of SMN on ribosomes, unravelling that SMN binds to both ribosomal subunits; ii) that SMN protein plays a key role in the translation elongation phase of translation; iii) that SMN organizes a translational platform of hundreds proteins, among which eIF3e; iv) that eIF3e and SMN proteins control a subset of mRNAs with rare codons at the beginning of the coding sequence and that show defects in ribosome occupancy at the start codon; v) that eIF3e sedimentation with the translation machinery is SMN-dependent and vi) that translation defects in mouse models of SMA appear at pre-symptomatic stage of disease and are mouse model-independent. To understand the impact of post-transcriptional heterogeneity in 2’-O-methylations in rRNA in SMA, I performed RiboMethSeq on total rRNA and on rRNA isolated from assembled ribosome of Taiwanese mouse brain and revealed a single defect in 2’-O-methylation level in position 18S:A578 on assembled ribosome in SMA, indicating that SMN loss also affects ribosome heterogeneity by altering 2’O-methylation pattern. Overall, this thesis shows that SMN is a fundamental protein in shaping ribosome heterogeneity and that its loss causes translational defects already at pre-symptomatic stages of the disease. A deeper molecular understanding of SMN role in translation will pave the way for the identification of translation-based SMA modifiers.